Transcription activator-like effector nucleases (TALENs) are fusions of the FokI restriction endonuclease cleavage domain with a DNA-binding transcription activator-like effector (TALE) repeat array. TALENs can be engineered to specifically bind and cleave a desired target DNA sequence, which is useful for the manipulation of nucleic acid molecules, genes, and genomes in vitro and in vivo. Engineered TALENs are useful in the context of many applications, including, but not limited to, basic research and therapeutic applications. For example, engineered TALENs can be employed to manipulate genomes in the context of the generation of gene knockouts or knock-ins via induction of DNA breaks at a target genomic site for targeted gene knockout through non-homologous end joining (NHEJ) or targeted genomic sequence replacement through homology-directed repair (HDR) using an exogenous DNA template, respectively. TALENs are thus useful in the generation of genetically engineered cells, tissues, and organisms.
TALENs can be designed to cleave any desired target DNA sequence, including naturally occurring and synthetic sequences. However, the ability of TALENs to distinguish target sequences from closely related off-target sequences has not been studied in depth. Understanding this ability and the parameters affecting it is of importance for the design of TALENs having the desired level of specificity and also for choosing unique target sequences to be cleaved, e.g., in order to minimize the chance of undesired off-target cleavage.